Teeth, the hard structures projecting from an anterior part of the alimentary tract and used for grasping and grinding food, vary from the chitinous projections on the radula of mollusks to the complex structures of the vertebrates. Vertebrate teeth are formed of two layers of hard materials over a living papilla, the pulp, which contains blood vessels and nerves. They form in the embryo from ingrowths of the outermost layer of the body, the ectoderm, associated with masses differentiated in the middle layer, the ectomesenchyme. The ectomesenchyme forms the dental papilla and around its outer surface, except at the end that is to remain connected with the body, lays down a layer of dentine. This material is similar to bone in composition but not in minute structure as it is harder and contains less organic matter than bone.
In human beings, beginning between the fifth and sixth months of fetal development, a layer of enamel is deposited over the dentine. Enamel is of ectodermal origin, being formed by protein secretions from ameloblasts, which are part of the enamel organ formed from an ingrowth of the epithelium of the oral ectoderm lining of the mouth. Enamel is made up of apatite and is the hardest substance prcduced by the animal's body, containing less than 1% organic matter. When the tooth takes its place in the jaw in the process of eruption, the enamel organ is destroyed, and no more of this substance can be produced. The enamel-producing cells in a human being (i.e. ameloblasts), are non-dividing cells, that is, they do not reproduce. The major function of ameloblasts is the production of enamel proteins, which in turn participate in the production of enamel.
Human teeth assume a fixed form. The only compensation for breakage and wear is found in the replacements that occur in the lower vertebrates. Mammals normally have no more than two sets of teeth, milk and permanent, and once the permanent teeth of adults have assumed their functional position in the jaw, loss through accident is permanent, as no new dental primordia or enamel can be formed.
The primary cause of tooth damage in humans is decay of the tooth, i.e. dental caries. Dental caries is the process in which bacteria adhere to the tooth surface, especially in pits and other harbored areas, to form plaques. Dental plaque comprises bacteria that are able to remain attached to the tooth surface because they secrete a sticky mucinous substance which is impervious to substances that might harm the bacteria. For example, water, mouthwash and saliva have little ability to penetrate the sticky mass, but fermentable carbohydrates penetrate easily. These foods are sources of energy for the dental caries-promoting bacteria.
These bacteria with their enzymes are capable of acting on fermentable foods to form acids. When sugar or carbohydrates contact dental plaque, acids are produced within a few minutes. The concentration increase continues, and by the end of a half-hour, the concentration may be sufficient to partially decalcify the tooth enamel. Decalcification stops when the acids are neutralized, until more fermentable sutstance is brought into the plaque, upon which the cycle is repeated. As the enamel demineralization continues, a hole or cavity is produced. When a cavity forms, the area becomes more difficult to clean and the microbes flourish.
The restoration of dental cavities through the surgical removal of decalcified enamel and the placement of dental restorative fillings is the most successful means of repairing a carious lesion. Silver amalgams, cast gold inlays, gold foil, quartz crystal composite resins and various organic polymers have served for years as effective agents for repair. Similar dental restorative materials are also used to fabricate entire teeth forms for dental replacement and/or repair. However, these artificial restorative materials are not without certain disadvantages, as are known in the art.
While the afore-mentioned dental restorative materials may produce workable dental reconstruction, metal replacement teeth and fillings tend to deteriorate, loosen or produce electrolytic action in the mouth, and organic polymers may require hazardous solvents or complicated mixing techniques. In addition, artificial repair materials tend to differ from the host structure (i.e., the natural tooth) with regard to coefficient of thermal expansion, and thus the ingestion of hot or cold foods or liquids tends to cause movement of the repair material relative to the tooth, and the eventual separation of the filling therefrom. These disadvantages result from the fact that the reconstruction materials heretofore used have artificial substances which have been used because tooth enamel is not produced by the body other than at selected times during development, and enamel production may not be selectively stimulated or produced externally. In other words, while broken bones are repaired by the body and the repair may be assisted by the proper setting of the fracture and bone growth stimulation by drugs, electricity or exercise, broken or damaged teeth may only be repaired by artificial means. It would be a substantial step forward in the art of dental science if teeth could be repaired or reconstructed with calcium hydroxyapatite crystals in a biosynthetic matrix form, i.e., natural tooth enamel. Heretofore, it has been possible to instigate crystal growth to form aprismatic matrices having various shapes and sizes. For example, calcium hydroxyapatite crystals have been grown from stable supersaturated calcium phosphate solutions by nucleation. However, it has not been possible to grow hydroxyapatite crystals in the form of dental prismatic enamel, this form being responsible for the physical properties of natural teeth.
The study of enamel proteins offers much information useful not only in basic research regarding tooth formation, but also in connection with the general aspects of craniofacial research and protein structure and function. However, such research has been limited due to the unavailability of enamel proteins in substantial quantities. Ameloblasts only produce enamel protein during a short period of time in mammals, and as the cells are non-dividing, they have not yet been cultured to produce the protein in vitro. Thus, the only method of obtaining enamel protein has been the post-mortem dissection of the forming enamel organic matrix from tooth organs of fetuses and neonates which is, of course, a limited supply. If enamel proteins were in greater supply, this problem would be overcome and basic research into the functionality and production of such proteins would be greatly accelerated. The ability to recover and synthesize the enamel protein DNA and RNA would be of great advantage in basic research and in the production of enamel protein, and the enamel protein may be used to grow apatite crystals in the form of natural dental enamel, e.g. as an advantageous dental restorative material.
The process of amelogenesis in vitro includes the biosynthesis and secretion of amelogenin and enamelin polypeptides from secretory ameloblasts upon a mineralizing dentine extracellular matrix. Both amelogenins and enamelins are glycosylated and phosphorylated. During the development of teeth when the enamel matrix is being formed, the total protein content comprises the initial enamel organic matrix. Mature enamel, in contrast to bone, has only 0.05 to 1.5% organic material on the basis of the weight of the enamel. Therefore, the biological strategy of enamel formation in vivo represents a relationship between the biochemical properties of the enamel extracellular organic matrix and subsequent biomineralization resulting in crystal nucleation, formation and growth.
Historically, enamel proteins were physically isolated on the basis of their solubility in a decalcifying solution [10% ethylenediaminetetraacetic acid (EDTA)]adjusted to pH 7.4. More recently it was shown by Termine and his colleagues J.Biol. Chem. 255:9760-9768 (1980)]that amelogenins were essentially soluble in guanidinium hydrochloride (4M), whereas enamelins were soluble in this dissociative solvent only with the addition of EDTA. It has been assumed that amelogenins provide a general structural constituent of the enamel matrix and also mediate the intracellular transport of amorphous calcium through the ameloblast cell for secretion to form the enamel matrix. During the transition phase from amorphous calcium phosphate to the crystallographic structure of enamel hydroxyapatite crystals [Ca.sub.10 (PO.sub.4)6(OH)2 with a calcium and phosphate (Ca/P)molar ratio of 1.67], the amelogenin polypeptides decrease in size and concentration whereas the enamelin polypeptide concentration remains somewhat constant. The unique chemical and physical properties of the mature enamel, therefore, are the resultant of numerous interactions between the different enamel proteins with appropriate concentrations of inorganic ions under physiological conditions.